Large-scale disintegration of microorganisms by freeze-pressing

A semicontinuous press has been constructed for the disintegration of microorganisms and other biological material by freeze-pressing, i.e., pressure extrusion of frozen material through a narrow hole. The material to be freeze-pressed is frozen in the form of cylindrical rods, which fit into the pressure chamber and are extruded by a piston forced back and forth by means of a hydraulic pump. At a sample temperature of ?35°C and a press temperature of ?20°C, about 90% disruption is achieved in a single passage of undiluted baker's yeast (Saccharomyces cerevisiae, 270 mg/g) through the orifice of the pressure chamber. With this press about 10 kg of material can be freeze-pressed per hour.     

Influence of cell concentration,temperature, and press performance on flow characteristics and disintegration in the freeze-pressing of Saccharomyces cerevisiae with the X-press

The pressure required for initiation of flow when freeze-pressing with the X-press is related to the phase boundaries of water, particularly those between ice I and liquid even at temperatures around ?25°C and lower. Widening the orifice of the pressure chamber to diameters larger than 2.5 mm leads to lower pressures and less extensive cell disintegration. Pressing Saccharomyces cerevisiae slowly with the aid of a manual hydraulic jack at ?25°C produces a disintegration of 60–75% irrespective of cell concentration. Pressing at ?35°C shows no clear differences. Pressing more rapidly with the aid of a motor-driven hydraulic press produces a similar extent of disruption of diluted cell suspensions (5.4 mg/g) as slow pressing. However, freeze-pressing a paste of baker's yeast (270 mg/g) increases the degree of disintegration. Under these conditions the disintegration is further enhanced by a lower temperature, ?35°C, and by a high velocity of flow through the orifice, such that more than 95% of the S. cerevisiae is disrupted by one pressing at less than 2 × 10⁸ Pa. Mechanisms for flow through the X-press are suggested and discussed in relation to the phase diagram of water.     

The contuation of the phase-boundary between ice I and liquid into the region of ice III and II and its relation to freeze-pressing of biological material.

The trajectory of the phase-boundary between ice I and liquid has been continuously followed by compression of deionized water, 0.10 m KCl, 0.10 m NaCl, and deionized water with suspended yeast cells (Saccharomyces cerevisiae, 180 mg/g) in a close-ended pressure chamber at temperatures below 0 °. Upon increasing pressure on deionized H₂O at ?8.6 °C the temperature first increases, until the transition line between ice I and liquid is reached. Then the sample cools on further compression, which is concomitant with an increase in electrical conductivity, indicating the gradual formation of liquid. At ?34.8 °C the pressure drops spontaneously from 3 × 10⁸ to 2.4 × 10⁸ Pa, the conductivity decreases, and the volume of the samples becomes further reduced to ?3.1 cm³/mole of H₂O, making the formation of ice III probable. On increase of pressure on 0.10 m KCl and 0.10 m NaCl the sample is gradually cooled, as the fusion line of the respective eutectic solid is reached. 0.10 m KCl is then super-cooled into the region of ice III and II, whereas 0.10 m NaCl is desalinated with a final conductivity of the suspension of 3–10 nmho/cm. In the sample with S. cerevisiae 180 mg/g the ice I-liquid phase-boundary was followed to ?36.0 °C into the region and ice III and II.These results are of great importance to the understanding of the freeze-pressing process, since they indicate that a transition from ice I to liquid may occur even at temperatures between ?22 °C and ?35 °C, thus facilitating flow of material through the press. This way they shed light on the pressures needed to initiate flow at different temperatures and compositions of the sample to be freeze-pressed.     

Influence of salts and gelatin on disintegration of Saccharomyces cerevisiae by freeze-pressing

The disintegration by freeze-pressing of a low concentration of Saccharomyces cerevisiae suspended in aqueous solutions of gelatin and different salts has been studied at different temperatures. In the freeze-pressing process deionized water and salt solutions flow in pulses, whereas samples with increasing concentrations of gelatin or cells tend to flow more smoothly. This smooth flow enhances the disruption efficiency particularly at lower temperatures, which seems to be of great practical importance. The addition of salts also promotes disintegration. The presence of both gelatin and salts works antagonistically on disintegration presumably because of different modes of action at disruption of cells.     

Tris buffer causes acyl chain interdigitation in phosphatidylglycerol

The structure of the gel phase and the properties of the acyl chain disordering transition of dipalmitoyl phosphatidylglycerol (DPPG) have been studied using differential scanning calorimetry, differential scanning dilatometry, and X-ray diffraction. In the presence of small, monovalent cations, DPPG at 22 degrees C exists in a lamellar phase in which the hydrocarbon chains are tilted from the perpendicular to the bilayer surface. Around 34 degrees C, there is a small pretransition (delta H less than 1 kcal/mol) followed by the main transition at 40.4 degrees C (delta H = 8.3 kcal/mol; delta V = 0.0381 ml/g). If DPPG is suspended in Tris-HCl buffer in the absence of other monovalent cations, X-ray diffraction data show that at 22 degrees C, the gel phase consists of interdigitated acyl chains perpendicular to the plane of the bilayer. No pretransition is observed and the main transition occurs at 41.3 degrees C with delta H = 9.1 kcal/mol and delta V = 0.0514 ml/g. If sufficient Na+ or K+ ions are added to the Tris-buffered DPPG, the phase behavior reverts to what is observed in the absence of Tris. Analysis of the energetics of the main transition shows that the increase in van der Waals interaction energy resulting from the larger delta V in Tris can be compensated by the favorable energetics of removing terminal methyl groups from the bilayer surface. The amount of disordering, i.e. formation of gauche rotamers, is likely to be the same in Tris as it is in buffers without amphiphilic cations.     

Ultrastructural studies of the mycelium-to yeast transformation of Sporothrix schenckii.

Fine details of the internal and external morphology of the in vitro mycelial phase (MP) to yeastlike phase (YP) transition of the dimorphic fungal pathogen Sporothrix schenckii are shown in electron micrographs of ultrathin sections. Morphological transformation at the ultrastructural level was observed to occur by direct formation of budlike structures at the tips and along the hyphae and by oidial cell formation. Direct budding of yeast from conidiospores was not observed. Early transitional forms arising by direct blastic action from the MP possessed conspicuous electron-dense microfibrillar material at the outer limits of the cell wall. The electron density of this microfibrillar material was enhanced by staining with acidified dialyzed iron. It is believed that this extracellular material may be composed in part of an acid mucosubstance. No acid phosphatase activity was associated with this microfibrillar material. This substance was found to be a characteristic of the outer limits of the cell wall of the YP of S. schenckii. Oidial YP cell formation occurred later during the transition. The cell wall of the developing oidial YP transitional form arose from an inner layer of the converting hyphae. No consupicuous alterations of the cytoplasmic content of the parent MP cell was observed during MP-to-YP transition. It is suggested that the MP-to-YP transition of S. schenckii may be regulated by at least two mechanisms involving alterations of the biochemical and/or biophysical nature of the cell wall of the MP cell in response to the conversional stimuli.     

STUDIES ON THE MAINTENANCE OF ORAL DEVELOPMENT IN TETRAHYMENA PYRIFORMIS GL-C : II. The Relationship of Protein Synthesis to Cell Division and Oral Organelle Development

The effects of puromycin on synchronized Tetrahymena pyriformis were investigated at two different concentrations, 43 µg per ml and 430 µg per ml. The rate of incorporation of histidine-¹⁴C into hot TCA-insoluble material was reduced by 30% at the low concentration and by 80–90% at the high concentration. The rate of oxygen uptake was lowered by only 10–20% at both concentrations. Cell division was prevented at both concentrations, if the drug was added prior to a "transition point" at about 45 min after the end of the synchronizing treatment. Development of "anarchic field" oral primordia was arrested, while primordia in early stages of membranelle differentiation were resorbed. Resorption began shortly after addition of the drug, and proceeded most rapidly at the lower concentration. If the drug was added after the "transition point," cell division and oral primordium formation were completed with only slight delay at the low concentration, and with considerable delay (in some cases complete arrest) at the high concentration. The results thus indicate that protein synthesis is involved in the later as well as the earlier stages of development; what specially characterizes the earlier stages, prior to the "transition point," is a dramatic response to partial inhibition of protein synthesis. It is suggested that this response involves the activation or release of a latent intracellular degradative system which is specific for developing structures.     

RNA synthesis in imaginal disks of Galleria mellonella: Effects of alpha- and beta-ecdysone and fat body in vitro

The effects of alpha-ecdysone (α-E), beta-ecdysone (β-E), and larval fat body on morphogenesis and total RNA synthesis in wing imaginal disks of Galleria mellonella were studied. Both ecdysones induce morphogenesis of disks in vitro. Alpha-ecdysone and β-E (0·3–3·0 μg/ml) stimulate RNA synthesis 30 and 60 per cent above control levels, respectively. While less α-E (0·3 μg/ml) is required to increase RNA synthesis than to induce morphogenesis, the reverse is true for β-E. Morphogenesis (i.e. tracheole migration and evagination) can proceed in the presence of concentrations of β-E (0·03 μg/ml) that are subthreshold for the induction of RNA synthesis (0·3 μg/ml). We conclude, therefore, that the increase in total RNA (presumbly ribosomal) is unrelated to and not a prerequisite for tracheole migration or evagination. If morphogenically active preconditioned medium (i.e. medium in which α-E and fat body have been incubated for 48 hr and the fat body then removed) is added to disk cultures, RNA synthesis is not stimulated. Apparently, increases in total RNA caused by both ecdysones may not be necessary for early in vitro disk development. The independent nature of some ecdysone-induced events and implications of our conclusions are discussed.     

Beta transition and stress-induced phase separation in the spinning of spider dragline silk

Spider dragline silk is formed as the result of a remarkable transformation in which an aqueous dope solution is rapidly converted into an insoluble protein filament with outstanding mechanical properties. Microscopy on the spinning duct in Nephila edulis spiders suggests that this transformation involves a stress-induced formation of anti-parallel beta-sheets induced by extensional flow. Measurements of draw stress at different draw rates during silking confirm that a stress-induced phase transition occurs.     

A ‘simple clock’ approach of circadian rhythms: An easy way to predict the clock's singularity

A graphical method is presented which allows the prediction of phase resetting curves of circadian rhythms for both type 1 and type 0 resetting, starting from one experimentally determined phase resetting curve. Calculations were based on literature data for the pupal eclosion rhythm of Drosophila pseudoobscura. The method is based on the assumption that for all practical purposes the rhythm may be approached as a “simple clock”, i.e. an oscillator with only one state variable, namely its phase or circadian time, CT. Besides predicting both “types” of phase resetting the method is capable to locate the “position” of the phase singularity and thus the transition from type 1 to type 0 resetting. This graphical method is an elaboration of the “transformation method”, developed in 1972 by A. Johnsson and H. G. Karlsson, which was effective in predicting phase resetting by “strong” stimuli, but failed in the case of “weak” stimuli. Predictions made using the extended transformation method are in good agreement with experimental results obtained with Drosophila. Also for the flesh fly, Sarcophaga argyrostoma, a prediction is made of the position of the phase singularity of the eclosion rhythm and compared with experimental results.     

A theoretical model of the temperature- and pressure-induced phase transition of phospholipid bilayers

A statistical thermodynamic model of phospholipid bilayers is developed. In the model, a new concept of a closely packed system is applied, i.e., a system of hard cylinders of equal radii, the radius being a function of the average number of gauche rotations in a hydrocarbon chain. Using this concept of a closely packed system, reasonable values are obtained for the change in specific volume at the order-disorder transition of lecithin bilayers. In addition to interactions between the lipid matrix and water molecules, between the head groups themselves and between hydrocarbon chains, as well as the intramolecular energy associated with chain conformation, the Hamiltonian of the membrane also includes the energy of the pressure field. Thus, the phase transition of phospholipid membranes induced not only by temperature hut also by hydrostatic pressure is described by this model simultaneously. In accordance with the experimental results, a linear relationship is obtained between the phase transition temperature and phase transition pressure. The other calculated phase transition properties of lecithin homologues. e.g., changes in enthalpy, surface area. thickness and gauche number per chain are in agreement with the available experimental data. The ratio of kink to interstitial conduction of bilayers is also estimated.     

High sensitivity differential scanning calorimetry of the bilayer to hexagonal phase transitions of diacylphosphatidylethanolamines

The bilayer to hexagonal phase transition of dioleoylphosphatidylethanolamine has been detected for the first time by differential scanning calorimetry. The observed transition is dependent on scan rate. This dependence can be explained by assuming that at rapid scan rates, the rate of conversion of bilayer to hexagonal phase is too slow at low temperatures for equilibration to take place. At higher temperatures the rate of interconversion becomes more rapid. The transition is observed to occur at 14°C using a scan rate of 0.74 K/min while it is centered at 8°C using a scan rate of 0.19 K/min. The enthalpy of the transition is 290 ± 40 cal/mol lipid and the transition is characterized by a ΔCp of −9 ± 1 mcal K⁻¹ (g lipid)⁻¹. The bilayer to hexagonal phase transition of dielaidoylphosphatidylethanolamine and of 1-palmitoy1-2-oleoylphosphatidylethanolamine occurs at 65.6°C and 71.4°C, respecitvely, with a corresponding transition enthalpy of 450 ± 20 and 400 ± 30 cal/mol lipid. The transitions of these phosphatidylethanolamines, occuring at higher temperatures, are independent of scan rate and show a higher degree of cooperativity than that of dioleoylphosphatidylethanolamine. Compared with the gel to liquid-crystalline transition of bilayer phospholipids the transition to hexagonal phase has a much lower enthalpy.     

Thermotropic behavior of retinal rod membranes and dispersions of extracted phospholipids

Summary High sensitivity, differential scanning calorimetry studies of vovine retinal rod outer segment (ROS) disk membranes and aqueous dispersions of the extracted ROS phospholipids have been performed. ROS disk membranes were found to exhibit a broad peak of excess heat capacity with a maximum at less than about 3°C, ascribable to a gel-to-liquid crystalline phase transition of traction of the phospholipids. A similar thermotropic transition was observed for aqueous dispersions of the total extracted and purified ROS phospholipids. Comparison of the results obtained for the dispersion of total ROS phospholipids to those of the purified head group fractions. suggests that the thermotropic behavior reffects a gel-to-liquid crystalline transition, leading to lateral phase separation, involving those phosphatidylcholine (PC) molecules containing saturated fatty acylchains, possibley together with the highest melting ROS phosphatidylethanolamine (PE) and phosphatidylserine (PS) components. The interpretation of the thermal behavior of the ROS disk membranes depends on whether the transition is assumed to derive from the ROS PC and/or PE/PS fractions, and whether the transbilayer arrangement of the ROS phospholipids is assumed to be symmetric or asymmetric. The calorimetric data can be simply explained in terms of an asymmetric distribution of the major ROS disk membrane phospholipids (G.P. Miljanich et al.,J. Membrane Biol. 60:249–255, 1981). In this case, the transition would arise from the PE/PS fractions in the outer ROS disk membrane monolyer, and the anticipated transition from the PC in the inner monolayer would be broadened due to interaction with cholesterol. For the ROS membranes at higher temperatures, two additional, irreversible transitions are observed at 57 and 72°C, corresponding to the thermal denauturation of opsin and rhodopsin, respectively.     

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.¹ Bacterial biofilms are encased within a matrix and attached to a surface.² Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)³ or growing biofilms for visualization (flow cells)⁴. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.⁵ The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.^6,7,8,9The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.¹⁰ The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.^9,11,12,13Download video file.^{(49M, mov)}     

Stable cupola-shaped bilayer lipid membranes with mobile Plateau-Gibbs border: expansion-shrinkage of membrane due to thermal transitions.

Stable bilayer lipid membranes (BLM) with mobile Plateau-Gibbs border (PGB) have been formed. The precondition of the formation was the presence of a lipid coverage on the teflon surface near the hole, where the membrane has been formed. This allowed the movement of the PGB along the teflon surface after transformation of the planar bilayer into a cupola-shaped by bowing of the bilayer due to excess hydrostatic pressure. As a result the giant bilayers were obtained with an area up to two orders larger in magnitude compared with the initial area. Changes in lipid bilayer area depend on the temperature at the phase transition of the lipid. Cooling of the expanded bilayer was followed by a significant shrinkage of the bilayer at temperatures below the main phase transition.     

Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom

The present article describes an l-amino acid oxidase from Bothrops atrox snake venom as with antiprotozoal activities in Trypanosoma cruzi and in different species of Leishmania (Leishmania braziliensis, Leishmania donovani and Leishmania major). Leishmanicidal effects were inhibited by catalase, suggesting that they are mediated by H₂O₂ production. Leishmania spp. cause a spectrum of diseases, ranging from self-healing ulcers to disseminated and often fatal infections, depending on the species involved and the host’s immune response. BatroxLAAO also displays bactericidal activity against both Gram-positive and Gram-negative bacteria. The apoptosis induced by BatroxLAAO on HL-60 cell lines and PBMC cells was determined by morphological cell evaluation using a mix of fluorescent dyes. As revealed by flow cytometry analysis, suppression of cell proliferation with BatroxLAAO was accompanied by the significant accumulation of cells in the G0/G1 phase boundary in HL-60 cells. BatroxLAAO at 25 μg/mL and 50 μg/mL blocked G0-G1 transition, resulting in G0/G1 phase cell cycle arrest, thereby delaying the progression of cells through S and G2/M phase in HL-60 cells. This was shown by an accentuated decrease in the proportion of cells in S phase, and the almost absence of G2/M phase cell population. BatroxLAAO is an interesting enzyme that provides a better understanding of the ophidian envenomation mechanism, and has biotechnological potential as a model for therapeutic agents.     

Influence of the bacterial growth phase on the magnetic properties of magnetosomes synthesized by Magnetospirillum gryphiswaldense

Background: The magnetosome biosynthesis is a genetically controlled process but the physical properties of the magnetosomes can be slightly tuned by modifying the bacterial growth conditions.Methods: We designed two time-resolved experiments in which iron-starved bacteria at the mid-logarithmic phase are transferred to Fe-supplemented medium to induce the magnetosomes biogenesis along the exponential growth or at the stationary phase. We used flow cytometry to determine the cell concentration, transmission electron microscopy to image the magnetosomes, DC and AC magnetometry methods for the magnetic characterization, and X-ray absorption spectroscopy to analyze the magnetosome structure.Results: When the magnetosomes synthesis occurs during the exponential growth phase, they reach larger sizes and higher monodispersity, displaying a stoichiometric magnetite structure, as fingerprinted by the well defined Verwey temperature. On the contrary, the magnetosomes synthesized at the stationary phase reach smaller sizes and display a smeared Verwey transition, that suggests that these magnetosomes may deviate slightly from the perfect stoichiometry.Conclusions: Magnetosomes magnetically closer to stoichiometric magnetite are obtained when bacteria start synthesizing them at the exponential growth phase rather than at the stationary phase.General significance: The growth conditions influence the final properties of the biosynthesized magnetosomes. This article is part of a Special Issue entitled “Recent Advances in Bionanomaterials” Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.